Methods for administering vaccines for aquaculture

Abstract

A method for delivering DNA vaccines to fish is provided, in which the vaccine is delivered intramuscularly in multiple injections.

Description

[Technical Field]

[0001] The present invention relates generally to the field of DNA vaccines for aquaculture. [Background technology]

[0002] Aquaculture has experienced significant growth in production, achieving over 527% growth between 1990 and 2018. In 2018, aquaculture contributed approximately 46% of the world's total aquatic production (179 million tonnes) and 52% of seafood (fish, crustaceans, mollusks, and other aquatic animals excluding aquatic mammals, reptiles, seaweed, and other aquatic plants) for human consumption.

[0003] Commercial aquaculture is affected by infectious diseases caused primarily by bacteria, viruses, parasites, and, to a lesser extent, fungi. Bacterial diseases can inflict significant biological and therefore economic losses. While they are usually controllable with antibiotics, the indiscriminate use of these medicines ultimately poses a threat to human health due to the development and transmission of resistance mechanisms among bacterial species, some of which are also human pathogens. Furthermore, some conventional vaccines based on inactivated viruses can cause unacceptable side effects, and / or some pathogens are difficult to grow in culture.

[0004] Pancreatic disease (PD) represents an economically important viral disease affecting Atlantic salmon (Salmo salar) farmed in seawater in Ireland, the UK, and Norway. The pathogen is officially named salmon pancreatic disease virus (SPDV), but because the virus also infects other salmonid species, it is commonly referred to as salmon alphavirus (SAV). Oil-adjuvanted vaccines containing inactivated whole virus particles of SPDV have been used for over a decade to mitigate the impact of PD on the salmonid aquaculture industry. However, in some cases, the use of these vaccines has resulted in serious side effects, including spinal deformities and fibrosis of surrounding muscle tissue, resulting in lower slaughter grades, reduced growth, and animal welfare concerns. See Baverfjord et al., NOFIMA report 35, 2021, and Holm et al., Aquaculture, 526, 2020.

[0005] Cardiomyopathy syndrome (CMS) is an inflammatory heart disease that primarily affects farmed Atlantic salmon, Salmo salar L. The disease was first detected in Norway in 1985 and has since been diagnosed in both farmed and wild Atlantic salmon. The causative agent of CMS has been identified as Piscine Myocarditis Virus (PMCV), whose isolation and characterization are described in International Publication No. WO 2011 / 131600. Methods for controlling the virus are urgently needed, but despite intensive research, PMCV has not been able to be efficiently cultured, nor has it been possible to produce a vaccine based on attenuated or inactivated virus.

[0006] DNA vaccines have become an attractive approach for generating antigen-specific immune responses due to their stability and simplicity of delivery: they can be easily prepared on a large scale with high purity, can be administered repeatedly, and are very stable compared to proteins and other biological polymers.

[0007] Among the many forms of nucleic acid vaccines that can be constructed, circular DNA plasmids are the simplest. DNA vaccination involves immunization with a circular DNA plasmid containing a gene (or genes) encoding an antigen. In fact, injection of free DNA (naked DNA) stimulates an effective and long-lasting immune response to the protein (antigen) encoded by the genetic vaccine. When plasmid DNA is injected into an individual, the plasmid is taken up by cells, and its genetic information is translated into the immunizing protein. This allows the host immune system to respond to the antigen.

[0008] However, DNA vaccines are relatively new and there is a need to improve the efficacy of these vaccines. Summary of the Invention

[0009] In one aspect, the present disclosure provides a vaccine for use in a method of actively protecting fish against a pathogen, the method comprising administering the vaccine to the fish intramuscularly in multiple injections, the vaccine comprising a nucleic acid sequence encoding an antigen derived from the pathogen affecting the fish, the method comprising administering the vaccine to the fish intramuscularly in multiple injections, a. Multiple components are delivered to different injection sites; b. each component of the plurality contains less than an effective dose of the vaccine; c. The total dose of the vaccine does not exceed the effective dose; d. The components are all delivered to the fish substantially simultaneously.

[0010] Also disclosed are vaccines as described above, wherein the components act synergistically.

[0011] 3. The vaccine of claim 1 or claim 2, wherein the total dose, when administered to fish kept under the same conditions, provides a more rapid onset of immunity than an effective dose administered in a single injection.

[0012] In certain embodiments of the vaccines made according to the methods described herein, the total dose of the vaccine is less than the efficacious dose.

[0013] In another aspect, the present disclosure provides a vaccine for use in a method for actively protecting fish against a pathogen that affects fish, the method comprising administering the vaccine to the fish intramuscularly in multiple injections, the vaccine comprising a nucleic acid sequence encoding an antigen derived from the pathogen; a. Multiple components are delivered to different injection sites; b. all of the components are delivered to the fish substantially simultaneously; The components act synergistically or the total dose administered in multiple injections, when administered to fish maintained under the same conditions, provides a more rapid onset of immunity than the same dose of the same formulation administered in a single injection. Preferably, the components act synergistically and the total dose administered in multiple injections, when administered to fish maintained under the same conditions, provides a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

[0014] Preferably, the plurality has 2, 3, or 4 components. The plurality can be delivered within 60 seconds, more preferably within 30 seconds, and even more preferably within 15 seconds.

[0015] Also disclosed is the above vaccine, wherein the fish is a salmonid and the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

[0016] In certain embodiments of the above vaccines, the pathogen is a salmon alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the pathogen is a PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments, the vaccine comprises both an antigen against a salmon alphavirus and an antigen against a PMCV.

[0017] Also disclosed is the above vaccine, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and tilapia lake virus.

[0018] Also disclosed are vaccines as described above, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector, such as a plasmid vector or a viral vector.

[0019] Also disclosed is the above vaccine, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments, the molecular immunomodulator is an interferon.

[0020] Also disclosed is the above vaccine, further comprising an adjuvant.

[0021] Also disclosed are the above vaccines, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane. In certain embodiments, the means comprises a lipid coating.

[0022] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) a reservoir containing the vaccine of any one of claims 1 to 20, the reservoir being operably connected to a plurality of needles configured to deliver components of a plurality of injections to different injection sites; b) means for applying pressure to the reservoir, thereby forcing the vaccine through the plurality of needles.

[0023] In yet another aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: a plurality of reservoirs, each reservoir containing components of one of multiple injections of the vaccine of any one of claims 1-21, each reservoir operably connected to a needle, wherein needles connected to different reservoirs are configured to deliver the components of the multiple injections to different injection sites; b. one or more means for applying pressure to the reservoir, thereby forcing the vaccine through the plurality of needles.

[0024] In both this and the previous aspect of the invention, the means may come from a number of sources, including, without limitation, pneumatic, hydraulic, electrical or mechanical sources.

[0025] In yet another aspect, the present disclosure provides a method of administering an effective dose of a nucleic acid-based vaccine to a fish, the method comprising delivering multiple subquantities of the effective dose to different injection sites in the fish, the multiple subquantities being injected substantially simultaneously.

[0026] In certain embodiments, in the methods disclosed herein, the plurality has two, three, or four components and can be delivered in 60 seconds, or 30 seconds, or 15 seconds or less. In certain embodiments, the plurality has two components and is delivered in 15 seconds or less. In other embodiments, the plurality has three components and is delivered in 15 seconds or less.

[0027] In certain embodiments of the methods described in this aspect, the fish is a salmonid and the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

[0028] In particular embodiments of the methods according to this aspect of the invention, the pathogen is a salmon alphavirus and the antigen comprises SEQ ID NO: 1 or SEQ ID NO: 2. In particular embodiments, the pathogen is PMCV and the antigen comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0029] Also disclosed is a method according to this aspect of the invention, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and tilapia lake virus.

[0030] In certain embodiments of the method according to this aspect of the invention, the nucleic acid sequence encoding the antigen is delivered by a heterologous vector. In certain embodiments, the heterologous vector is a plasmid vector. In other embodiments, the heterologous vector is a viral vector.

[0031] Also disclosed are methods according to this aspect of the invention, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator. In certain embodiments, the molecular immunomodulator is an interferon.

[0032] Also disclosed is a method according to this aspect of the invention, wherein the vaccine further comprises an adjuvant.

[0033] Also disclosed are methods according to this aspect of the invention, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane. In certain embodiments, the means comprises a lipid coating.

[0034] Also disclosed are methods according to this aspect of the invention in which multiple partial amounts of ingredients act synergistically.

[0035] Also disclosed are methods according to this aspect of the invention, wherein the total dose provides a more rapid onset of immunity than an effective dose administered in a single injection. DETAILED DESCRIPTION OF THE INVENTION

[0036] In order to better understand the present invention, the following definitions are provided.

[0037] The term "about" applied to a reference number refers to the reference number plus or minus 10 percent of that value.

[0038] The term "different injection sites" refers to injection points that are spaced far enough apart to allow for different skin penetrations upon injection at those injection points. For example, the injection sites may be at least 0.5 mm apart. In different embodiments, the injection sites are 0.5 to about 100 mm apart, or 0.5 to about 50 mm apart, or about 1 to about 40 mm apart, or about 3 to about 30 mm apart, or about 5 to about 25 mm apart, or about 5 to about 20 mm apart, or about 10 to about 20 mm apart, or about 10 mm to about 20 mm apart, or about 10 mm to about 30 mm apart, or about 10 mm to about They are 40 mm apart, or about 10 mm to about 50 mm apart, or about 10 mm to about 60 mm apart, or about 10 mm to about 70 mm apart, or about 10 mm to about 80 mm apart, or about 10 mm to about 90 mm apart, or about 20 mm to about 30 mm apart, or about 20 mm to about 40 mm apart, or about 20 mm to about 50 mm apart, or about 20 mm to about 70 mm apart, or about 30 mm to about 40 mm apart.

[0039] The term "effective dose" refers to the amount of antigen that, when administered as a single injection in a given formulation, provides the desired level of protection against the pathogen of interest.

[0040] The term "multiple ingredients acting synergistically" means a) refers to the ability of a total dose to elicit a greater protective immune response than the same formulation containing an effective dose, when that total dose is substantially equal to the effective dose; or b) When the total dose is less than the effective dose, it refers to the ability of that total dose to elicit the same protective immune response as the same formulation containing the effective dose.

[0041] The term "nucleic acid-based vaccine" refers to a vaccine in which the antigen is encoded by a nucleic acid sequence. The nucleic acid sequence encoding the antigen must enter a cell, replicate, and be expressed into the antigen. Nucleic acid-based vaccines include, but are not limited to, DNA vaccines and mRNA vaccines. DNA vaccines can be vectorized by a plasmid or a heterologous viral vector.

[0042] The term "substantially simultaneously" refers to the time interval between injections of sub-doses of an effective dose of a vaccine disclosed herein. The sub-doses are injected substantially simultaneously if the last sub-dosage of the vaccine is injected within 5 minutes, preferably within 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 15 seconds, 5 seconds, or 1 second of the first sub-dosage.

[0043] The term "substantially the same amount of antigen" in the context of a component of a plurality of injections refers to an amount of antigen in each component of the N plurality of injections that varies between 1 / 2N and 1.5 / N of the total dose of antigen. Thus, if there are two components of the plurality of injections, each component of the plurality should preferably contain 1 / 4 to 3 / 4 of the total dose. If there are three components of the plurality, each component of the plurality should preferably contain 1 / 6 to 1 / 2 of the total dose. In the most preferred embodiment, each component of the N plurality of injections should contain about 1 / N of the total dose of antigen. Thus, if N=2 (two components of the plurality of injections), each component of the plurality should contain about 1 / 2 of the total dose. If N=3 (three components of the plurality of injections), each component of the plurality should contain about 1 / 3 of the total dose.

[0044] The phrase "protected against a pathogen" refers to both the absence (or reduced level) of infection and the absence (or reduced level) of symptoms associated with infection. Where a protective titer against a given pathogen is known, protection against the pathogen also means that a statistically significant proportion of fish achieves the protective titer following vaccination as described herein.

[0045] The term "total dose" refers to the sum of the doses of antigen administered in each component of multiple injections. For example, but not limited to, if a vaccine is administered in three injections, and each injection contains 100 ng of a vector containing a nucleotide sequence encoding an antigen, the total dose is 300 ng.

[0046] The phrase "a total dose substantially equal to an effective dose" refers to a total dose that is at least 90% but not more than 100% of the effective dose. Conversely, the phrase "a total dose less than the effective dose" refers to a total dose that is less than 90% of the effective dose.

[0047] The term "the same protective immune response" refers to a response that is at least 95% but not more than 110% of the baseline, which is the immune response elicited by a single injection containing a dose of antigen equal to the total dose. Conversely, the term "greater than" refers to a response that is greater than 110% of the baseline, unless tissue damage is measured, in which case the term "greater than" refers to a response that is less than 95% of the baseline.

[0048] It is believed that administering the same amount of vaccine in multiple injections improves antigen uptake by host cells, thereby increasing antigen expression, sparing antigen dose and achieving substantially the same protective immune response as a larger antigen dose (i.e., an effective dose) administered in a single injection, and / or potentially improving the protective immune response when the total dose of vaccine administered in multiple injections is the same as the effective dose.

[0049] In a first broad aspect, the invention provides a vaccine for use in a method of protecting fish from infection by a pathogen, the vaccine comprising a nucleotide sequence encoding an antigen derived from the pathogen, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein components of the multiple injections are delivered to different injection sites, each component of the multiple contains less than an effective dose of the vaccine, the total dose of the vaccine does not exceed the effective dose, and all components of the multiple are delivered to the fish substantially simultaneously.

[0050] In a preferred set of embodiments, the administration of components in multiple injections provides a synergistic protective immune response that is greater than the protective immune response elicited by the same vaccine administered in a single effective dose when both groups of fish (the group treated with a single effective dose and the group treated according to the claims of the present disclosure) are kept under the same conditions. This can be important because fish are cold-blooded animals, and tank water temperature can be important. The temperature itself is less important than keeping the two groups under the same conditions.

[0051] Also disclosed are vaccines administered according to the above disclosed methods, wherein the total dose of the vaccine is less than the effective dose.

[0052] The present invention also provides a vaccine for use in a method for actively protecting fish from pathogens, the vaccine comprising a nucleic acid sequence encoding an antigen derived from a pathogen that affects fish, the method comprising administering the vaccine intramuscularly to the fish in multiple injections, wherein components of the multiple injections are delivered to different injection sites and the multiple components are all delivered to the fish substantially simultaneously, and the multiple components act synergistically or when administered to fish held under the same conditions, the total dose administered in the multiple injections provides a more rapid onset of immunity than the same dose administered in a single injection of the same formulation. Preferably, the multiple components act synergistically and when administered to fish held under the same conditions, the total dose administered in the multiple injections provides a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

[0053] The immune response can be measured in several ways, depending on the pathogen and knowledge in the art. For example, the titer of pathogen-specific antibodies may be determined. In other embodiments, the protective immune response may be measured by the percentage of animals that seroconvert. This endpoint is particularly suitable for pathogens for which protective titers have been established.

[0054] In other embodiments, mortality or survival rate may be a suitable endpoint for determining synergy. Virus counts may also be a suitable endpoint for judging synergy. Alternatively, the prevalence of infection (i.e., the ratio of virus-positive fish to total fish) in at least an organ at one or more time points may be a suitable endpoint. Immune response may also be measured by organ or tissue damage (or rather, the lack of organ or tissue damage) in response to the test. Synergy in at least one of these endpoints indicates a synergistic protective immune response.

[0055] Vaccines administered according to these methods, when administered to fish held under the same conditions, may result in a faster onset of immunity than the same vaccine administered as a single injection. The onset of immunity can be measured by the same methods as immune response, including, but not limited to, virus count, tissue damage, or the percentage of fish showing no viral infection. A faster immune response after testing in the group administered multiple injections compared to the group administered a single injection indicates a faster onset of immunity, even if, at later stages, the respective immune responses elicited by the single and multiple injections are less pronounced.

[0056] Vaccines suitable for use with the methods described herein are those in which a nucleic acid molecule encoding an antigen enters the host's cells and expresses the antigen. Such vaccines can be DNA vaccines or mRNA vaccines. Both types of such vaccines are known in the art.

[0057] mRNA-based vaccines against COVID-19 have been approved for use in humans. Thus, in certain embodiments, the vaccines comprise mRNA sequences of proteins derived from pathogens that cause disease in fish.

[0058] DNA vaccines are also known. At least one DNA vaccine, CLYNAV®, has been approved in Europe to protect salmon from pancreatic disease caused by salmon alphaviruses. CLYNAV® consists of a DNA plasmid (pUK-SPDV-poly2#1) dissolved in phosphate-buffered saline. It contains no adjuvants or preservatives. The quantitative and qualitative composition is appropriately defined as "pUK-SPDV-poly2#1 DNA plasmid encoding salmon pancreatic disease virus (SPDV) proteins," 5.1–9.4 μg / 0.05 ml dose (101.6–188.4 μg / ml).

[0059] Thus, in different embodiments of the DNA vaccine of the present invention, the nucleic acid sequence encoding the antigen is in a heterologous vector. Several heterologous vectors are suitable for the present invention and are known in the art. Vectors include, but are not limited to, viral vectors, plasmid vectors, and circular DNA vectors, also known as doggybone vectors.

[0060] Suitable heterologous virus vectors include but are not limited to alphaviruses such as SAV, rhabdoviruses such as VHSV and IHNV, paramyxoviruses such as ASP, adenoviruses, poxviruses such as salmonella poxvirus, etc.These viruses can be genetically modified to remove the viral genome part involved in replication.Therefore, the resulting virus will be suitable for infecting fish cells and producing antigen, but will not be pathogenic.

[0061] Suitable plasmids include, but are not limited to, pUC-based vectors, pVAX vectors, pcDNA vectors, NTC vectors. In a preferred set of embodiments, the vector is NTC9385R (Nature Technology Corporation) or a variant thereof.

[0062] In other embodiments, the relatively new "Doggybone" or DBDNA™ plasmids may be used as vectors. DBDNA™ plasmids and processes for making these plasmids are described in at least International Publication Nos. WO2018033730, WO2016034849, WO2019193361, WO2012017210, and WO2021161051.

[0063] The advantage of this approach is that the vector can be synthesized in a cell-free process, thus improving manufacturing efficiency. The cell-free process preferably involves template amplification via strand-displacement replication. This synthesis releases single-stranded DNA, which can then be copied into double-stranded DNA using a polymerase. Alternatively, strand displacement can be achieved by providing a DNA polymerase and a separate helicase. The replicative helicase can open the double-stranded DNA and facilitate the progress of the leading-strand polymerase. The resulting double-stranded DNA concatemers are enzymatically cleaved and ligated, thus forming a doggybone-shaped DNA construct. The doggybone vector consists of a telomerase recognition site, the desired construct (including the antigen-encoding sequence, promoter, and polyA site), and their complementary sequences. Thus, the complementary portions of the vector hybridize with each other to form a helix.

[0064] Several pathogens are suitable for use as antigen sources in the nucleic acid-based vaccines disclosed herein. In certain embodiments, the pathogen is a virus. Suitable viruses may be selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), piscine orthoreovirus (PRV), lumpfish virus, viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and tilapia lake virus. In certain embodiments, the virus is a salmon alphavirus. In another embodiment, the virus is PMCV.

[0065] In other embodiments, the pathogen is a bacterium, including, but not limited to, Pissirickettsia, Aeromonas, Vibrio, Listonella, Moritella viscosa, Photobacterium damselae, Flavobacterium, Yersinia, Renibacterium, Streptococcus, Lactococcus, Leuconostoc, Bifidobacterium, Pediococcus, Brevibacterium, Edwardsiella, Francisella, Pseudomonas, Cytophaga, Nocardia, and Mycobacerium.

[0066] Particularly preferred are antigens from intracellular bacterial pathogens, including, but not limited to, Pissirickettsia, Edwardsiella, Yersinia, Francisella, Photobacterium, Mycobacterium, and Renibacterium.

[0067] Surface proteins from viruses and bacteria may be suitable candidates for the nucleic acid-based vaccines disclosed herein.

[0068] In other embodiments, the pathogen is a parasite, such as, for example, a sea louse. In certain embodiments, the sea louse is selected from the genus Lepeophtheirus or Pisces, and the antigen can be a midgut protein or an immunogenic fragment thereof. See, e.g., U.S. Patent No. 11,167,017.

[0069] Certain vaccines particularly suitable for administration to salmonid fish, such as Salmo salar, contain nucleic acid sequences encoding immunogens that elicit a protective immune response against salmon alphaviruses. Suitable non-limiting examples of such antigens include, but are not limited to, SEQ ID NO: 1 or SEQ ID NO: 2.

[0070] Other vaccines suitable for administration to salmonid fish, such as Salmo salar, include nucleic acid sequences encoding immunogens that elicit a protective immune response against piscine myocarditis virus (PMCV). Suitable, non-limiting examples of such antigens include the protein encoded by ORF-1 of PMCV (e.g., SEQ ID NO: 3), or fragments thereof (including, but not limited to, SEQ ID NO: 4).

[0071] Additionally, other vaccines suitable for administration to Salmo salar, etc., include nucleic acid sequences encoding immunogens that induce a protective immune response against piscine myocarditis virus (PMCV) and immunogens that induce a protective immune response against salmon alphaviruses, as described above.

[0072] In another embodiment, the pathogen is a parasite, such as a sea lice, particularly of the genus Lepeophtheirus, more particularly of the species Lepeophtheirus salmonis. Peptides derived from sea lice have been proposed as potential antigens for vaccines. Some suitable candidates are disclosed in U.S. Patent No. 11,167,017.

[0073] The nucleic acid sequence of a DNA vaccine is generally under the control of a suitable promoter. Suitable promoters for vaccines according to the present invention must be capable of initiating transcription in the host organism. In embodiments in which the host is a salmonid fish, such as Salmo salar, suitable promoters include, but are not limited to, the simian virus 40 early promoter (SV40), the cytomegalovirus immediate early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF1A), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken β-actin promoter (CAGG) linked to the CMV early enhancer.

[0074] The vector may further comprise a nucleic acid sequence encoding a molecular immunomodulator. Suitable molecular immunomodulators include interferons. It has previously been demonstrated that nucleic acid sequences encoding salmon interferon delivered by DNA vaccines enhance antigen-specific immune responses. Thus, in certain embodiments, the molecular immunomodulator is an interferon selected from the group consisting of salmon IFNa, IFNb, IFNb1, IFNa2, and IFNc.

[0075] The vaccine may also contain an adjuvant. Suitable adjuvants include, but are not limited to, saponin (e.g., Quil A), alum, CpG oligonucleotides, oligoribonucleotides, cytokines, glycolipids such as BAY® 1005, and quaternary amines such as dimethyldioctadecylammonium bromide (hereinafter, "DDA"). Complexes containing saponin, sterol (e.g., cholesterol), and optionally, phospholipids have been described in the art. Combinations of CpG oligonucleotides and saponin, CpG and cholesterol, and CpG and alum have been reported to induce synergistic effects.

[0076] In certain embodiments, the vaccine may include a liposomal adjuvant and / or carrier to facilitate transport of the vector across the cell membrane, thus resulting in increased expression of the antigen and / or molecular immunomodulator. Suitable non-limiting examples of such liposomal adjuvant / carrier systems are described, for example, in U.S. Patent No. 10,456,459.

[0077] The vaccine according to the present invention may further comprise excipients such as preservatives, stabilizers, buffers, etc.

[0078] Several fish species are suitable for vaccination according to the methods described herein. Suitable species include salmonids (including species of the genus Salmon and Salmon), as well as sea bass (Dicentrarchus labrax), and warmwater fish including tilapia (Oreochromis niloticus) and pangasius (Pangasius Hypophthalmus). Other species include whitefish, Arctic char, mandarin perch, and largemouth bass. Fish can be vaccinated according to the methods disclosed herein when the fish weigh between about 15 grams and about 200 grams, more preferably between about 40 grams and about 110 grams.

[0079] As mentioned above, the vaccine according to the present invention is delivered by multiple injections in several portions administered substantially simultaneously (the first and last portion being administered within 5 minutes of each other), preferably the last portion is administered within 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 15 seconds, 5 seconds, and 1 second of the first portion of the vaccine.

[0080] The exact number of components in the multiple injections is generally 2, 3, 4, or 5, more preferably 2 or 3.

[0081] The volumes of the components can be determined by one of skill in the art, but generally, the volumes can be independently selected from 0.01 ml to about 0.25 ml, including about 0.02 ml, about 0.025 ml, about 0.05 ml, about 0.075 ml, about 0.1 ml, about 0.15 ml, about 0.2 ml, or 0.25 ml. It is currently preferred that the components of the multiple injections contain substantially the same amount of antigen.

[0082] There are several ways to administer injections substantially simultaneously. In certain embodiments, commercially available fish vaccination devices can be fitted with a multi-needle injection tip, with the needle tips configured to be a desired distance from one another. Suitable vaccination machines include the NFTT product line (Pharmaq), with specific models including the NFT20, NFT25, and NFT30. The NFT20 and NFT25 deliver vaccines intraperitoneally but can also be reconfigured for intramuscular injection. The NFT30 NFT30 has a special DNA module that allows for intramuscular administration of DNA vaccines into fish fillets.

[0083] The machine handles fish ranging in size from 120mm to 250mm (20-150 grams). When finished, it sorts the vaccinated fish into three different sizes. In addition, there are tubes for misplaced, undersized or rejected fish.

[0084] Alternatively, vaccines can be administered manually using a syringe with one or more needles. See the MICRO-MATIC® syringes sold by Pharmaq. This product is available in single and double sizes. The single syringe is available in two sizes: 0.05 ml per dose and 0.1 ml per dose. Additionally, the 0.05 ml syringe can be equipped with an interchangeable 0.025 ml piston if desired. The double syringe is available in three different dose size combinations: 0.05 ml + 0.05 ml per dose, 0.05 ml + 0.1 ml per dose, and 0.1 ml + 0.1 ml per dose. The 0.05 ml syringe can be equipped with an interchangeable 0.025 ml piston if desired. These syringes can be used for both aqueous and oil-based vaccine formulations. The dose size can be easily adjusted within + / - 10%. Dose accuracy has been documented to be less than 3%.

[0085] The pressure used to deliver the injection is not critical and can be derived from hydraulic, pneumatic, electrical, or mechanical sources.

[0086] Generally, the exact number of injections (i.e., components of a plurality of injections) can be determined empirically. Preferably, the number is between 2 and 5, more preferably between 2 and 4, and most preferably 2 or 3.

[0087] In one embodiment, there are three components of the multiple injections, and the antigen comprises a PMCV antigen, such as SEQ ID NO:3 or SEQ ID NO:4. In these embodiments, the vaccine may also contain an immunomodulator, such as an interferon. Preferably, the interferon is IFNb. In another embodiment, there are two components of the multiple injections, and the antigen comprises a PMCV antigen, such as SEQ ID NO:3 or SEQ ID NO:4. In these embodiments, the vaccine may also contain an immunomodulator, such as an interferon. Preferably, the interferon is IFNb.

[0088] In another embodiment, there are two components of the multiple injections and the antigen comprises an SAV antigen, for example, SEQ ID NO:1 or SEQ ID NO:2.

[0089] The following examples are presented as illustrative embodiments and should not be construed as limiting the scope of the present invention. Many changes, variations, modifications, and other uses and applications of the present invention will be apparent to those skilled in the art. [Example]

[0090] Example 1. Multiple injections of SAV DNA vaccine result in lower viral loads and clinical scores than a single injection. Materials and Methods: Atlantic salmon juveniles weighing an average of 26 grams were vaccinated in freshwater by intramuscular vaccination with a standard dose of PD DNA vaccine. One group received a single 0.05 ml dose. The other group received two consecutive 0.025 ml injections of 0.05 ml each. Fish injected with phosphate-buffered saline (PBS) served as a negative control. Fish were housed in the same tank throughout the study. Beginning the day after vaccination, fish were exposed to continuous light to induce smoltification and produce the physiological changes necessary to prepare the fish for transfer to seawater. After a 6-week (42 days, 500 degree-days) immunization period, fish were transferred to seawater and challenged with SAV3 in a symbiotic challenge. The challenge was performed by intraperitoneally injecting infectious SAV3 material into naive fish, which were then introduced into the same tank to expose vaccinated and control fish to SAV3 in a manner that mimicked the occurrence of natural infection. Twenty-nine to 30 vaccinated fish were tested per group, and 20 negative control fish were tested per group. All surviving fish were collected at the end of the coexistence test, 5 weeks (36 days) after the start of the test. Fish were weighed, and heart and pancreas samples (both important target organs of PD) were collected using formalin and RNALATER®. No mortality occurred during the observation period in the two vaccinated groups, and only one mortality (5%) occurred in the negative control fish. Therefore, efficacy was assessed primarily by assessing body weight, the severity of tissue damage to the heart and pancreas, and the amount of SPDV in the heart and pancreas.

[0091] Statistical analysis of cardiac and pancreatic viral loads in vaccinated groups compared to the negative control group was performed by the Mann-Whitney test using GRAPHPAD PRISM® v.8.1.1. Statistical analysis of end-of-study body weights in vaccinated groups compared to the negative control group was performed by an unpaired t-test with Welch's correction using GRAPHPAD PRISM® v.8.1.1.

[0092] Quantification of SPDV in cardiac and pancreatic tissue samples was performed by a probe-based reverse transcriptase quantitative PCR (RT-qPCR) assay. This assay targets SPDV nonstructural protein 1 (nsP1) and can detect all known salmon alphaviruses (Hodneland & Endresen, 2006). Analysis was performed by Pharmac Analytic, an officially accredited diagnostic laboratory authorized in Norway to officially validate SPDV infection in salmonids.

[0093] At the end of the 5 weeks post-test (wpc), surviving fish were analyzed for the severity of tissue damage by histopathology on a scale of 0 to 3 (pancreas) and 0 to 4 (heart) using the scoring system described by Graham et al., Journal of Fish Diseases, vol. 34, issue 4, pp. 237-286, 2011. This scoring system is shown in Table 1. [Table 1]

[0094]

[0093] The results are shown in Table 2 (virus count), Table 3 (severity of tissue damage), and Table 4 (body weight). [Table 2]

[0095] PCR results show that both the single and double injection groups reduced the prevalence of infection and reduced viral load (increased numbers), but prevalence was lower in the two-injection group. Heart viral loads in both the single and double-injection groups were significantly different from controls. Pancreatic viral loads were significantly different from controls in the two-injection group. These results indicate that two 0.025ml injections of vaccine are more effective than a single 0.05ml injection of vaccine. [Table 3]

[0096] The tissue damage in the pancreas and heart of fish in the single-injection and double-injection groups was different from that of the control group, respectively. Notably, in both the pancreas and heart, the double-injection group tended to show less damage than the single-injection group (0.13 vs. 0.41 in the heart, 0.17 vs. 0.31 in the pancreas). [Table 4]

[0097]

[0096] Body weights of vaccinated groups 5 weeks after the start of the coexistence study indicate that both vaccinated groups were highly protected against PD-related growth reduction, with significantly higher body weights and growth rates compared to the negative control group. No statistically significant differences were observed between the single- and double-injection groups.

[0098] Increasing the number of injections from one to two decreased the viral load and reduced viral prevalence in both the heart and pancreas 5 weeks into the study. Despite the total amount of vaccine being the same in both groups, the prevalence in both the heart and pancreas was reduced by more than two-fold in the two-injection group compared to the single-injection group.

[0099] Example 2. Multiple injections of PMCV DNA vaccine result in lower viral loads and lower histopathology scores than single injections. Atlantic salmon (n = 150), weighing an average of 29 g, were housed in 500 L tanks containing freshwater (12C) under a 12:12 light:dark photoperiod. After fasting for 1 day, the fish were anesthetized using MS222 (tricaine, PHARMAQ AS), tagged in one of five groups (n = 30 per group) by shortening the adipose fin or maxilla, and intramuscularly vaccinated with a DNA plasmid expressing a partial ORF1 PMCV antigen and salmon IFNb as a molecular adjuvant. One of the five groups received a negative control vaccine (PBS), while the remaining four groups received one, two, three, or four injections of a vaccine containing the PMCV ORF1 coding sequence under the same anesthesia period. Plasmid concentrations were adjusted for each group to achieve a total plasmid load of 10 μg per fish, regardless of the number of injections. Table 5 shows group details at the time of vaccination. [Table 5]

[0100] After 7 weeks of immunization, all fish were anesthetized again and intraperitoneally injected with 0.1 ml of kidney homogenate containing infectious PMCV particles. Hearts were then harvested from 15 fish per group per time point at 3 and 7 weeks after the study. The hearts were stored in RNALATER® and sent to the diagnostic laboratory PHARMAQ Analytiq (Bergen, Norway) for real-time PCR detection of PMCV RNA. Hearts harvested 7 weeks after the study were also examined for cardiac pathology (PHARMAQ Analytiq, Bergen, Norway). The virus counts at 3 and 7 weeks after the study are summarized in Tables 6 and 7, respectively. Histological analysis of the atria is summarized in Table 8. [Table 6] [Table 7] [Table 8]

[0101] Increasing the number of injections from one to three resulted in an increase in virus counts (a decrease in viral load) three weeks after the test, as well as an increase in the percentage of fish found to be negative (from two in the group treated with one injection to eight in the group treated with three injections). Seven weeks after the test, the number of negative fish was 5 out of 15 in the groups treated with one, two, and four injections, and 7 out of 15 in the group treated with three injections. These results suggest that vaccines administered in multiple injections result in a rapid onset of immunity. No significant differences in body weight were observed between the groups treated with one, two, three, and four injections.

[0102] All publications, both patent and non-patent, cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0103]

[0102] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications can be made to the exemplary embodiments and that other arrangements can be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. 1. A vaccine for use in a method for actively protecting fish against a pathogen that affects fish, the vaccine comprising a nucleic acid sequence encoding an antigen derived from said pathogen, the method comprising administering the vaccine to said fish intramuscularly in multiple injections; a. the components are delivered to different injection sites; b. each component of the plurality contains less than an effective dose of the vaccine; c. the total dose of the vaccine does not exceed the effective dose; d. A vaccine wherein all of said components are delivered to said fish substantially simultaneously.

2. The vaccine of claim 1 , wherein the plurality of components act synergistically.

3. 3. The vaccine of claim 1 or claim 2, wherein the total dose, when administered to fish kept under the same conditions, results in a more rapid onset of immunity than the effective dose administered in a single injection.

4. 1. A vaccine for use in a method for actively protecting fish against a pathogen that affects fish, the vaccine comprising a nucleic acid sequence encoding an antigen derived from said pathogen, the method comprising administering the vaccine to said fish intramuscularly in multiple injections; a. the components are delivered to different injection sites; b. all of the components are delivered to the fish substantially simultaneously; A vaccine wherein the components act synergistically or when administered to fish kept under the same conditions, the total dose administered in the multiple injections results in a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

5. 5. The vaccine of claim 4, wherein the components act synergistically and when administered to fish kept under the same conditions, the total dose administered in the multiple injections results in a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

6. The vaccine of any one of claims 1 to 5, wherein the total dose of the vaccine is less than the effective dose.

7. The vaccine of any one of claims 1 to 6, wherein the plurality comprises 2, 3 or 4 components.

8. The vaccine of any one of claims 1 to 7, wherein the multiple injections are delivered within one minute.

9. The vaccine of any one of claims 1 to 8, wherein the multiple injections are delivered within 30 seconds.

10. 10. The vaccine of any one of claims 1 to 9, wherein the multiple injections are delivered within 15 seconds.

11. The vaccine of any one of claims 1 to 10, wherein each component of the plurality contains substantially the same amount of antigen.

12. a. the plurality has two components, each component of the plurality having about half the total dose of the antigen; or b) The vaccine of claim 11, wherein said plurality has three components, each component of said plurality having about 1 / 3 of said total dose of said antigen.

13. 13. The vaccine of any one of claims 1 to 12, wherein the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), piscine orthoreovirus (PRV), lumpfish virus, viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), tilapia lake virus, Pissirickettsia, Edwardsiella, Yersinia, Francisella, Photobacterium, Mycobacterium, Renibacterium, Lepeophtheirus, and Piscipida.

14. 13. The vaccine of any one of claims 1 to 12, wherein the fish is a salmonid and the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

15. a) the plurality has two or three components and the pathogen is PMCV; or b) A vaccine according to claim 13 or 14, wherein the plurality has two components and the pathogen is SAV.

16. 14. The vaccine of any one of claims 1 to 13, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and tilapia lake virus.

17. The vaccine of any one of claims 1 to 16, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector.

18. The vaccine of claim 17, wherein the heterologous vector is a plasmid vector or a viral vector.

19. 19. The vaccine of claim 17 or 18, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator.

20. 20. The vaccine of claim 19, wherein the molecular immunomodulator is an interferon.

21. The vaccine of any one of claims 1 to 20, further comprising an adjuvant.

22. 22. The vaccine of claim 21, wherein the adjuvant is selected from the group consisting of MPLA, CpG-containing oligodeoxyribonucleotides, oligoribonucleotides, saponins, sterols, and cationic lipids.

23. 23. The vaccine of any one of claims 1 to 22, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane.

24. 24. The vaccine of claim 23, wherein the means comprises a lipid coating.

25. a) a reservoir containing the vaccine of any one of claims 1 to 24, said reservoir operatively connected to a plurality of needles, said plurality of needles configured to deliver the components of said plurality of injections to different injection sites; b) means for applying pressure to said reservoir, thereby forcing said vaccine through said plurality of needles; 1. An article of manufacture comprising:

26. a plurality of reservoirs, each reservoir containing a component of a plurality of injections of the vaccine of any one of claims 1-24, each reservoir operatively connected to a needle, wherein needles connected to different reservoirs are configured to deliver the components of the plurality of injections to different injection sites; b. one or more means for applying pressure to said reservoir, thereby forcing said vaccine through said plurality of needles; 1. An article of manufacture comprising:

27. A method for administering an effective dose of a nucleic acid-based vaccine to a fish, the method comprising delivering multiple sub-doses of the effective dose to different injection sites of the fish, the multiple sub-doses being injected substantially simultaneously.

28. 28. The method of claim 27, wherein the components act synergistically.

29. 29. The method of claim 27 or 28, wherein multiple sub-doses of the effective dose are injected into the fish at different injection sites, resulting in a more rapid onset of immunity when administered to fish maintained under the same conditions than when the effective dose is administered as a single injection.

30. A method of administering a total dose of a nucleic acid-based vaccine to fish, comprising delivering multiple sub-doses of the total dose to different injection sites on the fish, wherein the multiple sub-doses are injected substantially simultaneously, and wherein the components act synergistically or the total dose administered in multiple injections, when administered to fish held under the same conditions, results in a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

31. 32. The method of claim 31 , wherein the components act synergistically or the total dose administered in the multiple injections, when administered to fish held under the same conditions, results in a more rapid onset of immunity than the same dose of the same formulation administered in a single injection.

32. The method of any one of claims 27 to 31, wherein the total dose is less than the effective dose of the same vaccine.

33. 33. The method of any one of claims 27 to 32, wherein the plurality comprises 2, 3 or 4 components.

34. 34. The method of any one of claims 27 to 33, wherein the multiple subdoses are delivered within one minute.

35. 35. The method of any one of claims 27 to 34, wherein the multiple sub-doses are delivered within 30 seconds.

36. 36. The method of any one of claims 27 to 35, wherein the multiple sub-doses are delivered within 15 seconds.

37. 37. The vaccine of any one of claims 27 to 36, wherein each component of the plurality contains substantially the same amount of antigen.

38. a. the plurality has two components, each component of the plurality having about half the total dose of the antigen; or b) The vaccine of claim 37, wherein said plurality has three components, each component of said plurality having about 1 / 3 of said total dose of said antigen.

39. 39. The method of any one of claims 27 to 38, wherein the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), piscine orthoreovirus (PRV), lumpfish virus, viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), tilapia lake virus, Pissirickettsia, Edwardsiella, Yersinia, Francisella, Photobacterium, Mycobacterium, Renibacterium, Lepeophtherius, and Piscipida.

40. 40. The method of any one of claims 27 to 39, wherein the fish is a salmonid and the pathogen is selected from the group consisting of salmon alphavirus (SAV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (INHV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia (ISA) virus (ISAV), piscine myocarditis virus (PMCV), and piscine orthoreovirus (PRV).

41. a) the plurality has two or three components and the pathogen is PMCV; or 41. The method of claim 39 or 40, wherein b) the plurality has two components and the pathogen is SAV.

42. 40. The method of any one of claims 27 to 39, wherein the fish is tilapia and the pathogen is selected from the group consisting of viral nervous necrosis virus (NNV), infectious spleen and kidney necrosis virus (ISKNV), and tilapia lake virus.

43. The method of any one of claims 27 to 42, wherein the nucleic acid sequence encoding the antigen is delivered by a heterologous vector.

44. 44. The method of claim 43, wherein the heterologous vector is a plasmid vector or a viral vector.

45. 45. The method of claim 43 or 44, wherein the heterologous vector further comprises a nucleic acid sequence encoding a molecular immunomodulator.

46. 46. ​​The method of claim 45, wherein the molecular immunomodulator is an interferon.

47. The method of any one of claims 27 to 46, further comprising an adjuvant.

48. 48. The method of claim 47, wherein the adjuvant is selected from the group consisting of MPLA, CpG-containing oligodeoxyribonucleotides, oligoribonucleotides, saponins, sterols, and cationic lipids.

49. 49. The method of any one of claims 27 to 48, wherein the vaccine comprises a means for transporting the nucleic acid-based vaccine across a cell membrane.

50. 50. The method of claim 49, wherein the means comprises a lipid coating.

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